William C. Faquin, MD, PhD Director, Head & Neck Pathology Professor of Pathology Massachusetts Eye & Ear Massachusetts General Hospital Harvard Medical School

THE MILAN SYSTEM AND MOLECULAR ADVANCES IN THE CYTOLOGIC DIAGNOSIS OF SALIVARY GLAND TUMORS Salivary Gland FNA

Even if you do not adopt the Milan System in your practice, reviewing the structure of a reporting system will provide insight to salivary gland FNA! Salivary gland tumors are one of the most heterogeneous groups of neoplasms – So what role is there for FNA? Salivary Gland FNA-

4 Salivary Gland FNA – Pleomorphic Adenoma

5 Salivary Gland FNA-

6 Salivary Gland FNA- Solid Adenoid Cystic Carcinoma

7 SALIVARY GLAND FNA: How Far Can We Go?

Usually Specific Diagnosis Sometimes Specific Diagnosis Usually Descriptive Diagnosis

Pleomorphic adenoma Adenoid cystic carcinoma Basal cell adenoma, tubulotrabecular and solid types Warthin tumor LG HG mucoepidermoid carcinoma Acute and chronic Carcinoma ex PA Salivary duct carcinoma Basal cell adenoma, Metastasis Polymorphous low-grade membranous type Reactive lymph node Small cell carcinoma Basal cell adenocarcinoma Lymphoma Mucocele Epithelial-myoepithelial carcinoma Lymphoepithelial cyst MASC LESA SOME SALIVARY GLAND FACTS

• Tumors: • 0.4-13.5 per 100,000 people (uncommon) • Older adults, females, • Approx. 75% are benign • Risk of malignancy is inversely proportional to the size of the gland (20% in parotid; 80-89% in oral cavity)

SALIVARY GLAND FNA

Part of the reason for the high accuracy: A majority of SG neoplasms are pleomorphic adenomas, Warthin tumor, or metastatic cancer

Effectiveness of Cytomorphology alone: » Sensitivity: 86-100% » Accuracy: » Benign/low grade vs HG malignant: 81-98% » Specific lesion: 48-94%

SALIVARY GLAND FNA

Rationale for FNA: –Guide the clinical management/pre-op strategy: » Non-neoplastic Clinical follow-up » Benign tumor/low-grade carcinoma Limited resection » Metastatic disease to parotid LNs LN resection » Lymphoma Heme-Onc referral » High-grade primary carcinoma Radical resection Reporting System for Salivary Gland FNA

Why do we need a new reporting system for salivary gland cytology? Salivary Gland FNA

Diagnostic Terminology • Current reporting confusion: – Diversity of diagnostic categories, vs. – Descriptive reports (no categories), vs. – Surgical pathology terminology • General agreement on the need for a defined set of diagnostic categories for salivary gland FNA – Clarity of communication (implicit cancer risk) – Exchange of data across institutions • The Milan System for Reporting Salivary Gland Cytopathology The Milan System for Reporting Salivary Gland Cytopathology

• Sponsored by the ASC and the IAC • Over 40 participants from 14 countries • Goal is to produce a practical classification system that is user-friendly and internationally accepted. • The system will be evidence-based. • Print Atlas available in 2017 • Web-Based Atlas will also be available through the ASC Participants: 40+ Members from 14 Countries Cytopathologists, Surgical Pathologists, Molecular Pathologists, ENT Surgeons

• 1. Overview of Diagnostic Terminology and Reporting: – Core group, Bruce Wenig, Raja Seethala, Andrew Field, Nora Katabi • • 2.Non-diagnostic: – Mariapia Foschini (lead), Laszlo Vass, Esther Diana Rossi, Jhala Nirag, Philippe Vielh, Kayoko Higuchi, , Ivana Kholova, Makato Urano • • 3. Non-neoplastic: – Bill Faquin (lead), Massimo Bongiovanni, Sule Canberk, Marc Pusztaszeri, Tarik Elsheik, Dan Kurtycz, Fabiano Callegari, Oscar Lin • • 4. AUS: – Marc Pusztaszeri (lead), Zubair Baloch, Bill Faquin, Diana Rossi • • 5. Neoplastic (benign & SUMP): – Zubair Baloch (lead), Jeff Krane, Lester Layfield, Marc Pusztaszeri, Jerzey Klijanienko, Ritu Nayar, Celeste Powers, Pinar Firat, Guido Fadda • • 6.Suspicious for malignancy: – Diana Rossi (lead), Syed Ali, Ashish Chandra, Zarha Maleki, Bo Ping, He Wang, Andrew Field, Yun Gong, • • 7.Malignant: – Güliz Barkan (lead), He Wang, Philippe Vielh, Stefan E. Pambuccian, Swati Mehrotra, Mousa Al-Abbadi, Eva Wojcik • • 8. Ancillary Studies: – Mark Pusztaszeri (lead), Jorge Reis-Filho, Fernando Schmitt, Raja Seethala • • 9. Clinical Management: .. – Mark Varvares (lead –MGH), Piero Nicolai (Italy), Mandeep Bajwa The Milan System for Reporting Salivary Gland Cytopathology Diagnostic Categories

• 1) Non-Diagnostic • 2) Non-Neoplastic • 3) Atypia of undetermined significance • 4) Neoplastic: – a) Benign – b) Uncertain malignant potential • 5) Suspicious for Malignancy • 6) Malignant

The Milan System for Reporting Salivary Gland Cytopathology

The ROM will depend upon the nature of the Specimen and the salivary gland site.

18 Non-Diagnostic

• Insufficient quantitative and/or qualitative cellular material to make a cytologic diagnosis. • 10% would be a target maximum rate • Includes aspirates with benign elements only • Includes non-mucinous cyst contents Non-Diagnostic: Benign salivary gland elements only NORMAL SALIVARY GLAND FNA

For aspirates containing only normal salivary gland elements, a cautionary note is recommended. Note: Clinical and radiologic correlations are recommended to ensure that the aspirate is representative of the lesion; the findings in this aspirate do not explain the presence of a salivary gland mass. Non-Diagnostic: Non-mucinous cyst contents DDX: Ductal cyst, pseudocyst, cystic neoplasm

Absence of an epithelial component Non-Neoplastic

• Specimens lacking evidence of a neoplastic process: • Inflammatory, metaplastic, and reactive (includes acute, chronic, and granulomatous sialadenitis, sialadenosis, etc…) • Reactive lymph nodes (flow cytometry is needed) •Clinico-radiological correlation is essential to ensure that the specimen is representative of the lesion. Non-Neoplastic: Reactive Lymph Node Mixed population of lymphs, Tingible body macrophages, germ center frags REACTIVE PROCESS VS LYMPHOMA

• IMMUNOPHENOTYPING combined with cytomorphology is the key to diagnosing and subtyping reactive conditions vs lymphoma. For negative lymph nodes, caution is warranted: A note suggesting repeat FNA or tissue biopsy if lymphadenopathy persists. Non-Neoplastic: Chronic Sialadenitis

Hypocellular, cohesive basaloid groups, inflammation Non-Neoplastic: Granulomatous Sialadenitis

DDX includes infection, sarcoidosis, and neoplasm Non-Neoplastic: Atypia of Undetermined Significance (AUS)

• Cannot entirely exclude a neoplasm. • Heterogeneous category • A majority will be reactive atypia or poorly sampled neoplasms. • Specimens are often compromised (eg, air-drying, blood clot). • Should be used rarely (<10 % of all salivary gland FNAs).

Atypia of Undetermined Significance (AUS): Mucinous Cyst Contents Only- Cannot exclude MEC Atypia of Undetermined Significance (AUS): Oncocytic metaplasia vs Neoplasm Atypia of Undetermined Significance (AUS): Reactive vs basaloid neoplasm

Sclerosing polycystic adenosis Neoplasm

i) Benign Neoplasm: Reserved for clear-cut benign neoplasms This category will include classic cases of PA, WT, lipoma, etc… ii) Salivary Gland Neoplasm of Uncertain Malignant Potential: Diagnostic of a neoplasm; however, a diagnosis of a specific entity cannot be made. A malignant neoplasm cannot be excluded. Neoplastic: Benign Warthin Tumor

Oncocytes, chronic inflammation, and cystic debris Neoplastic: Benign Pleomorphic Adenoma

Matrix-rich types of PA are the easiest. Neoplastic: SUMP Pleomorphic Adenoma With Atypia or Metaplasia

Squamous metaplasia Focal atypia Neoplastic: SUMP Myoepithelioma Neoplastic: SUMP Basal Cell Neoplasm- DDX basal cell adenoma, cellular PA, AdCC Suspicious for Malignancy

• Aspirates which are highly suggestive of malignancy but not definitive. • Often high grade carcinomas with limited sampling or other limitation

Suspicious for Malignancy AdCC with Artifact Malignant

• Aspirates which are diagnostic of malignancy. • Sub-classify into specific types and grades of carcinoma: e.g. low grade vs high grade. • "Other" malignancies such as lymphomas, sarcomas and metastases are also included in this category and should be specifically designated. Malignant

Salivary Duct Carcinoma Undifferentiated Carcinoma Malignant: Adenoid Cystic Carcinoma Malignant: Mucoepidermoid Carcinoma Malignant: HG B-Cell Lymphoma Salivary Gland FNA

• Improvements in IHC and molecular testing will make the Milan System and salivary gland FNA in general more effective. • It is critical that the FNA specimen include adequate material for ancillary studies in difficult cases. Ancillary Studies to Improve the FNA Diagnosis of Head and Neck Tumors

 Immunocytochemistry  LBP  Smears  Cell block  FISH  RT-PCR  Next Generation Sequencing

NGS-SNaPshot Panel at MGH

• Anchored Multiplex PCR (AMP)

• ~190 target amplicons across 39 genes and 50+ rearrangements

• High-quality sequence: - Staggered start sites - >100X target coverage - Molecular indexing - Bi-template coverage - ~2% analytical sensitivity

• Fast turn-around (~2 weeks)

• Cost-effective (<$500)

• Small tissue amounts (5-10 ng)

NORMAL SALIVARY GLAND Immunohistochemical Markers

SOX10

Ker 7, 19, CAM 5.2, EMA Ker 7, CAM 5.2, DOG1

Ker 5/6, S-100, p63, calponin, SM actin Ker 5/6, p63 Increasing Availability of Molecular Markers For Salivary Gland Tumors

 Mammary analogue secretory carcinoma:  ETV6-NTRK3; t(12:15)  Pleomorphic adenoma & Ca ex PA:  PLAG1; t(3;8)  HMGA2 rearrangement  Clear cell carcinoma:  EWSR1-ATF1; t(12:22)  Mucoepidermoid carcinoma:  MECT1/MAML2; t(11:19)  Cribriform Adenocarcinoma:  PRKD rearrangement  Adenoid cystic carcinoma:  MYB-NFIB; t(6:9)  Basal cell adenoma:  CTNNB1 mutations

IHC and Molecular Analysis Applied to Selected Salivary Gland Tumors Pleomorphic Adenoma Diagnostic Problems Arise from Variants of PA

• Cellular PA with sparse matrix

• Focal adenoid cystic–like areas

• Cytologic atypia

• Metaplasia – Squamous

– Mucinous

– Sebaceous

– Oncocytic Pleomorphic Adenoma with Mucinous Metaplasia: Can be difficult to distinguish from MEC Pleomorphic Adenoma Immunohistochemistry:

• Markers of both ductal and myoepithelial cells: • Keratin 7, CEA, EMA, SOX10

– Myoepithelial markers:

• Smooth muscle actin

• Calponin

• S-100

• Keratin 5/6

• P63

• GFAP Pleomorphic Adenoma

Cytogenetics: • PLAG1 rearrangements (50-60%) – Present in PAs in different tissues – Nuclear localization – Functions to activate transcription (Zn finger) • HMGA2 rearrangements (10%)

http://www.uniprot.org/uniprot/Q6DJT9 PLAG-1 Immunoreactivity: Overexpressed in 94% of PA Does not distinguish benign from malignant

Contributed by Dr. J. Krane, BWH Carcinoma Ex Pleomorphic Adenoma: Most cases have PLAG1 or HMGA2 Rearrangements

Salivary duct carcinoma ex PA Adenoid Cystic Carcinoma Adenoid Cystic Carcinoma: Classic Cribriform Pattern Adenoid Cystic Carcinoma Immunohistochemistry:

• Positive for keratin 7, CEA, EMA

• Positive for myoepithelial markers: – Smooth muscle actin

– Calponin

– S-100

– Keratin 5/6

– P63

• SOX10+

• CD117 (KIT) +

• MYB + Adenoid Cystic Carcinoma

CD117 Immunohistochemistry:

• Over 90% are strongly positive for CD117 (KIT)

• Protein overexpression but no mutation identified

• Useful for all variants including solid forms

• Unusual IHC pattern Immunoreactivity for CD117 (KIT) in AdCC:

Tubular Type Solid Type

Luminal Cells + MYB immunostaining is a useful ancillary test for distinguishing adenoid cystic carcinoma from pleomorphic adenoma in FNA specimens

FNA BIOPSY Adenoid Cystic Carcinoma: Recent Advance – MYB Translocation Cytogenetics: • t(6:9) MYB oncogene-NFIB transcription factor • In salivary gland, this finding by FISH is specific for AdCC

FISH contributed by Dr. Joaquin Garcia, Mayo Clinic Basal Cell Adenoma/Adenocarcinoma Basal Cell Adenocarcinoma – Cytologically indistinguishable from adenoma Basal Cell Adenoma & Adenocarcinoma

Immunohistochemistry:

• Positive for keratin 7, CEA, EMA

• Positive for myoepithelial markers

• Nuclear beta-catenin + (esp. common in adenoma) Basal Cell Adenoma & Adenocarcinoma • CTNNB1 mutation – 3p21 • Beta-Catenin overexpression – Present at cell junctions – Part of WNT signaling pathway Nuclear Beta-Catenin in Basal Cell Adenoma Jo et al. AJSP 2016;40:1143-1150. Mammary Analogue Secretory Carcinoma FNA of Mammary Analogue Secretory Carcinoma MASC Immunohistochemistry:

• Positive for: – S-100

– Mammaglobin

– Keratin 7, 8, 19

– EMA

– GCDFP-15

– MUC1, MUC4

• Negative for: – Myoepithelial markers (p63 etc)

– Androgen receptor

– DOG1 +/- MASC: Immunohistochemical Studies

GCDFP-15+ S-100+

Mammoglobin+ Mammary Analogue Secretory Carcinoma: Cytogenetics: For difficult cases, test for rearrangement

 ETV6-NTRK3 rearrangement:

 T(12:15)(p13;q25)

 MASC is only known salivary gland primary

 Detected on histology or cytology using:

 FISH

 RT-PCR

 Next-Gen Sequencing

FISH Contributed by Dr. Joaquin Garcia, Mayo Clinic Acinic Cell Carcinoma Acinic Cell Carcinoma DOG1 and Acinic Cell Carcinoma

Discovered on GIST

Calcium-activated chloride channel protein

Strong diffuse staining seen in most acinic cell carcinomas SOX-10 and Acinic Cell Carcinoma SRY-related HMG-box 10 (SOX10) protein

Transcription factor: neural crest formation, and maintenance of Schwann cells and melanocytes.

Acinar cells and intercalated ducts

Diffuse strong nuclear staining seen in most acinic cell carcinomas SOX10 and Salivary Gland Tumors POSITIVE

Acinic cell carcinoma

Adenoid cystic carcinoma

Epithelial-myoepithelial carcinoma

Myoepithelial carcinoma

Pleomorphic adenoma

NEGATIVE

Salivary duct carcinoma

Mucoepidermoid carcinoma

Oncocytoma

Warthin tumor MUCOEPIDERMOID CARCINOMA Low Grade Mucoepidermoid Carcinoma: Hypocellular with few groups of bland epidermoid cells; Common cause of FN FNA Mucoepidermoid Carcinoma: IHC is non-specific Immunohistochemistry:

• Positive for: – Keratin 5,6,7,8,19

– EMA

– CEA

– p63

• Negative for: – SM actin

– Calponin

– S-100

– SOX10 Mucoepidermoid Carcinoma: Can be useful for difficult cases

Cytogenetics: • t(11:19) translocation • MECT1/MAML2 • FISH or NGS • More common in low grade • Seethala et al Am J Surg Pathol (2010) – 60% overall – LG 74%, IG 63%, HG 32% – Useful in small biopsies

FISH contributed by Dr. Joaquin Garcia, Mayo Clinic SUMMARY

• Salivary gland cytology presents many diagnostic challenges • The Milan System for Reporting Salivary Gland Cytoplathology will help to produce a more uniform diagnostic structure – Improved communication between treating clinician and pathologist – Improved patient care • Availability of IHC and molecular markers can greatly improve the accuracy of salivary gland FNA! The Milan System for Reporting Salivary Gland Cytopathology

We look forward to implementing the Milan system – Thank You!